Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2015 / Diabetes mellitus is one of the major diseases worldwide that is life threatening and is reaching an epidemic proportion. The most important approach in reducing the burden of the disease worldwide is to search for effective, low cost hypoglycaemic drugs with fewer side effects. Past experimental evidence confirmed the hypoglycemic activity of many indigenous African medicinal plants. S. italica (Fabaceae family) is widely used by traditional healers to treat a number of diseases such as sexually transmitted diseases and other forms of intestinal complications traditionally. The current study was aimed at evaluating the in vitro effects of root and leaf extracts of S. italica on GLUT4 translocation in NIH-3T3-L1 preadipocytes and C2C12 muscle cells. In order to address the aim of the study various methods were undertaken. The roots and leaves of S. italica collected from Zebediela sub-region of the Limpopo province, South Africa, were ground to fine powder and extracted using acetone, methanol, ethyl acetate and n-hexane. The various extracts of the root and leaf material were subjected to fingerprint profiling using TLC plates and different mobile phases (BEA, CEF, EMW and BAW). The chromatograms were visualized with vanillin-H2SO4 reagent, p-anisaldehyde and iodine vapour. The extracts were assayed for the type of secondary metabolites contained in the studied plant parts using chemical text and by TLC analysis. The total phenolic content of the root and leaf material were also evaluated. Evaluation for antioxidant activity was performed using 0.2% DPPH qualitatively and quantitatively with vitamin C as a positive control. Toxicity study was performed on C2C12 muscle cells using the MTT assay, with Curcumin as a positive control and untreated cells as a negative control. The CC50 values of the acetone root and leaf extracts were determined by linear regression. The effect of acetone root and leaf extracts on glucose uptake by C2C12 muscle cells was evaluated, also on western blot and immunofluorescence for NIH-3T3-L1 preadipocytes. The solvents employed for extraction in this study are commonly used to extract various biological active compounds from plants in research settings. Methanol extracted more compounds followed by acetone, then ethyl acetate and n-hexane the least. The constituents extracted by methanol may be mostly sugars, amino acids and glycosides due to the polarity of this solvent. Hydro-alcoholic solvents extract a variety of compounds that are mostly polar. Acetone extracts xxii mostly alkaloids, aglycones and glycosides while n-hexane in general extracts mostly waxes, fats and fixed oils. High yield was obtained with leaf extracts with all the solvent used for extraction as compared to the root. The TLC finger-print showed that good separation was achieved with the methanol and acetone extracts in CEF mobile phase, ethyl acetate extracts in CEF and EMW and n-hexane extracts in BEA respectively, especially with the leaf extract. Most compounds present in S. italica extracts were UV active. Some compounds that were not reactive with vanillin-H2SO4 reagent were shown to be reactive with p-anisaldehyde reagent and iodine vapour which revealed the presence of sugars or aromatic compounds. Chemical analysis for secondary metabolites of the acetone root and leaf extracts revealed the presence of flavonoids, terpenes, tannins, steroids, reducing sugars and alkaloids while glycosides were detected only in the leaf extract. The results obtained using TLC analyses were consistent with the results obtained in the chemical analysis. Thin layer chromatography revealed the presence of glycoflavones in the acetone root extract, alkaloids in the root and leaf extracts; and phytosterols and flavonoid aglycones in root and leaf extracts. The acetone root and leaf extracts revealed the presence of phenols. The leaf extract was shown to contain high total phenolic content as compared to the root. The methanol and acetone root and leaf extracts were shown to possess antioxidant activity. However, the concentration of the activity was higher in the acetone root than in the leaf extract. The least activity was observed with the ethyl acetate root and leaf extracts as compared to other extracts. The n-hexane extracts however, was not shown to contain any antioxidant compounds. Although activity observed with the methanol extracts was comparable to that of the acetone extracts in the quantitative assay, the acetone extracts were shown to possess more antioxidant activity in the qualitative assay. The concentration of extracts increased with increase in scavenging activity. The root extract exhibited a more potent antioxidant activity compared to leaf extract. These extracts were evaluated for their cytotoxicity on normal cells. The highest cytotoxic concentration (CC50) was obtained with the root extract with a CC50 value of 297 635 μg/ml at 48 hrs, followed by CC50 value of 21 544 μg/ml at 24 hrs. The CC50 value of the leaf extract at 24 hrs was 2 904 μg/ml with the least value at 48 hrs. The root extract at 24 and 48 hrs together with the leaf extract at 24 hrs were not toxic to C2C12 muscle cells at the concentration tested in this study. The acetone extracts were shown to possibly enhance proliferation of C2C12 muscle cells at a concentration of 0.001–1000 μg/ml. The non-cytotoxic concentration of 25 μg/ml of the leaf extract in combination with insulin showed more glucose uptake as compared to other extracts as well as the control. Prolonged incubation time was shown to increase glucose uptake with leaf extract while increase in concentration of root extract decreased glucose uptake at 24 hrs. At incubation time of 3 and 24 hrs, glucose uptake results at concentration of 2.5 μg/ml were comparable with that of the root extract, with a similar trend observed at 25 μg/ml, although with decrease in uptake. The qualitative and quantitative fluorescence results showed GLUT4 to be translocated to the cell membrane. The leaf extract at a concentration of 25 μg/ml had more fold as compared to other extracts, indicative that more GLUT4 was translocated at this concentration of the leaf extract. The acetone root and leaf extracts were shown to increase protein expression of GLUT4 at 3 hrs incubation time as compared to other incubation times in insulin-stimulated C2C12 muscle cells. The plant constituents of S. italica was shown to contain a variety of secondary metabolites that maybe be acting alone or in concert with each other to exert the various activities observed in this study. Different solvents used for extraction may be responsible for the extraction of different constituents with antioxidant activity observed in the study. The acetone extracts enhanced proliferation of C2C12 muscle cells at concentrations used in the study. However, there was no significant reduction on viability of normal cells. In addition, the extracts were shown to enhance the differentiation of NIH-3T3-L1 preadipocytes into adipocytes and C2C12 muscle cells into myocytes. These in turn induced the translocation of GLUT4 to the cell membrane and as a consequence facilitate glucose transport. Hence, the differentiation of adipose cells as well as glucose uptake of muscle cells and GLUT4 expression might have been enhanced by constituents contained in the acetone extracts. In conclusion, the acetone leaf extract may have a beneficial role in glucose metabolism of differentiated C2C12 muscle cells. Therefore, further studies are however required to elucidate the molecular mechanism by which the acetone leaf extract of S. italica influences the translocation of GLUT4.

The increasing reliance on drugs from natural sources has led to the development of several drugs from traditional plants which are present in abundance in Southern Africa. With the rapid increase of incidence of type 2 diabetes in South Africa with potentially devastating effects on healthcare, the need for alternative therapeutics is a priority. In this study, Schkuhria pinnata (Lam.) Kuntze was investigated for its antidiabetic potential. Initial screening of two different solvent extracts of S. pinnata identified an aqueous extract that lowered blood glucose concentrations in a hyperglycaemic streptozotocin-induced diabetic rat. The classical bioassay approach was followed by using different solvents, drying processes and fractionation in order to produce the most active extract and attempt to isolate an active compound(s). An aqueous freeze dried extract was found to be the most active at stimulating glucose uptake activity in C2C12 and Chang cells. Fractionation of this extract in an attempt to identify the active compound yielded a novel crystalline compound 1 by NMR analysis. Screening for bioactivity of the extract and compound 1 using C2C12 muscle and Chang cells revealed that both extract and compound 1 were biologically active, however the activity of the aqueous extract was more significant overall. A butanone/pentane extract prepared for possible commercialization purposes was also shown to be active in vitro. To establish antidiabetic activity, the aqueous freeze dried extract, butanone/pentane extract and the enriched compound 1 fraction were tested in a streptozotocin (STZ) diabetic rat model showing hypoglycaemic effects for the aqueous freeze dried extract. Messenger RNA and protein studies on C2C12 muscle cells revealed that the aqueous freeze dried extract and compound 1 enhanced insulin receptor, GLUT-4, glycogen synthase, pyruvate kinase and pyruvate carboxylase expression, suggestive of an insulin mimetic mode of action, while the butanone/pentane extract enhanced adenosine monophosphate-activated kinase (AMPK) protein expression by a non-insulin dependent mechanism. A pharmacokinetic study (PK) established bioavailability of compound 1 following oral administration of the extracts, but not from the compound 1 enriched fraction. From this study, the traditional use of S. pinnata has been scientifically validated as having antidiabetic properties. In vitro and in vivo bioassays, confirmed that an aqueous freeze dried extract which was prepared as per the traditional method had the most promising antidiabetic iii activity. Compound 1 isolated from an active fraction was proven to be almost as effective as the parent extract in in vitro studies. This compound could therefore be the major active ingredient responsible for the uptake of glucose in cells and the hypoglycaemic activity in vivo. In this study, the antidiabetic activities together with the mechanism of action of S. pinnata extracts and compound 1 were elucidated. The highlight of the study was the identification of a bioactive novel chemical entity (NCE) compound 1 (identified as 2-(2-{[(2E)-4-hydroxy2-(hydroxymethyl)but-2-enoyl]oxy}-4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)prop-2- enoic acid) isolated from an active fraction of S. pinnata that was proven to be almost as effective as the parent extract in in vitro studies. This compound could therefore be the major active ingredient responsible for the uptake of glucose in cells and the hypoglycaemic activity in vivo. The cellular mechanism of action of the S. pinnata extracts and compound 1 demonstrated both insulin mimetic and non-insulin dependent mechanisms (AMPK) in C2C12 muscle cells. Further research in the form of preclinical and clinical trials need to be undertaken to make this extract or biologically active compound available as a herbal remedy or nutraceutical therapeutic for diabetes. To achieve this; safety, efficacy and mode of action studies will have to be established. The synthesis of compound 1 and/or analogues should also be investigated as an antidiabetic drug candidate.

Thesis (M. Sc. (Biochemistry)) -- University of Limpopo, 2015 / Diabetes mellitus is one of the major diseases worldwide that is life threatening and is reaching an epidemic proportion. The most important approach in reducing the burden of the disease worldwide is to search for effective, low cost hypoglycaemic drugs with fewer side effects. Past experimental evidence confirmed the hypoglycemic activity of many indigenous African medicinal plants. S. italica (Fabaceae family) is widely used by traditional healers to treat a number of diseases such as sexually transmitted diseases and other forms of intestinal complications traditionally. The current study was aimed at evaluating the in vitro effects of root and leaf extracts of S. italica on GLUT4 translocation in NIH-3T3-L1 preadipocytes and C2C12 muscle cells. In order to address the aim of the study various methods were undertaken. The roots and leaves of S. italica collected from Zebediela sub-region of the Limpopo province, South Africa, were ground to fine powder and extracted using acetone, methanol, ethyl acetate and n-hexane. The various extracts of the root and leaf material were subjected to fingerprint profiling using TLC plates and different mobile phases (BEA, CEF, EMW and BAW). The chromatograms were visualized with vanillin-H2SO4 reagent, p-anisaldehyde and iodine vapour. The extracts were assayed for the type of secondary metabolites contained in the studied plant parts using chemical text and by TLC analysis. The total phenolic content of the root and leaf material were also evaluated. Evaluation for antioxidant activity was performed using 0.2% DPPH qualitatively and quantitatively with vitamin C as a positive control. Toxicity study was performed on C2C12 muscle cells using the MTT assay, with Curcumin as a positive control and untreated cells as a negative control. The CC50 values of the acetone root and leaf extracts were determined by linear regression. The effect of acetone root and leaf extracts on glucose uptake by C2C12 muscle cells was evaluated, also on western blot and immunofluorescence for NIH-3T3-L1 preadipocytes. The solvents employed for extraction in this study are commonly used to extract various biological active compounds from plants in research settings. Methanol extracted more compounds followed by acetone, then ethyl acetate and n-hexane the least. The constituents extracted by methanol may be mostly sugars, amino acids and glycosides due to the polarity of this solvent. Hydro-alcoholic solvents extract a variety of compounds that are mostly polar. Acetone extracts mostly alkaloids, aglycones and glycosides while n-hexane in general extracts mostly waxes, fats and fixed oils. High yield was obtained with leaf extracts with all the solvent used for extraction as compared to the root. The TLC finger-print showed that good separation was achieved with the methanol and acetone extracts in CEF mobile phase, ethyl acetate extracts in CEF and EMW and n-hexane extracts in BEA respectively, especially with the leaf extract. Most compounds present in S. italica extracts were UV active. Some compounds that were not reactive with vanillin-H2SO4 reagent were shown to be reactive with p-anisaldehyde reagent and iodine vapour which revealed the presence of sugars or aromatic compounds. Chemical analysis for secondary metabolites of the acetone root and leaf extracts revealed the presence of flavonoids, terpenes, tannins, steroids, reducing sugars and alkaloids while glycosides were detected only in the leaf extract. The results obtained using TLC analyses were consistent with the results obtained in the chemical analysis. Thin layer chromatography revealed the presence of glycoflavones in the acetone root extract, alkaloids in the root and leaf extracts; and phytosterols and flavonoid aglycones in root and leaf extracts. The acetone root and leaf extracts revealed the presence of phenols. The leaf extract was shown to contain high total phenolic content as compared to the root. The methanol and acetone root and leaf extracts were shown to possess antioxidant activity. However, the concentration of the activity was higher in the acetone root than in the leaf extract. The least activity was observed with the ethyl acetate root and leaf extracts as compared to other extracts. The n-hexane extracts however, was not shown to contain any antioxidant compounds. Although activity observed with the methanol extracts was comparable to that of the acetone extracts in the quantitative assay, the acetone extracts were shown to possess more antioxidant activity in the qualitative assay. The concentration of extracts increased with increase in scavenging activity. The root extract exhibited a more potent antioxidant activity compared to leaf extract. These extracts were evaluated for their cytotoxicity on normal cells. The highest cytotoxic concentration (CC50) was obtained with the root extract with a CC50 value of 297 635 μg/ml at 48 hrs, followed by CC50 value of 21 544 μg/ml at 24 hrs. The CC50 value of the leaf extract at 24 hrs was 2 904 μg/ml with the least value at 48 hrs. The root extract at 24 and 48 hrs together with the leaf extract at 24 hrs were not toxic to C2C12 muscle cells at the concentration tested in this study. The acetone extracts were shown to possibly enhance proliferation of C2C12 muscle cells at a concentration of 0.001–1000 μg/ml. The non-cytotoxic concentration of 25 μg/ml of the leaf extract in combination with insulin showed more glucose uptake as compared to other extracts as well as the control. Prolonged incubation time was shown to increase glucose uptake with leaf extract while increase in concentration of root extract decreased glucose uptake at 24 hrs. At incubation time of 3 and 24 hrs, glucose uptake results at concentration of 2.5 μg/ml were comparable with that of the root extract, with a similar trend observed at 25 μg/ml, although with decrease in uptake. The qualitative and quantitative fluorescence results showed GLUT4 to be translocated to the cell membrane. The leaf extract at a concentration of 25 μg/ml had more fold as compared to other extracts, indicative that more GLUT4 was translocated at this concentration of the leaf extract. The acetone root and leaf extracts were shown to increase protein expression of GLUT4 at 3 hrs incubation time as compared to other incubation times in insulin-stimulated C2C12 muscle cells. The plant constituents of S. italica was shown to contain a variety of secondary metabolites that maybe be acting alone or in concert with each other to exert the various activities observed in this study. Different solvents used for extraction may be responsible for the extraction of different constituents with antioxidant activity observed in the study. The acetone extracts enhanced proliferation of C2C12 muscle cells at concentrations used in the study. However, there was no significant reduction on viability of normal cells. In addition, the extracts were shown to enhance the differentiation of NIH-3T3-L1 preadipocytes into adipocytes and C2C12 muscle cells into myocytes. These in turn induced the translocation of GLUT4 to the cell membrane and as a consequence facilitate glucose transport. Hence, the differentiation of adipose cells as well as glucose uptake of muscle cells and GLUT4 expression might have been enhanced by constituents contained in the acetone extracts. In conclusion, the acetone leaf extract may have a beneficial role in glucose metabolism of differentiated C2C12 muscle cells. Therefore, further studies are however required to elucidate the molecular mechanism by which the acetone leaf extract of S. italica influences the translocation of GLUT4.

Résumé : Introduction : Le débat sur l’utilisation des hypoglycémiants oraux (HGO) dans le traitement du diabète gestationnel (DG) perdure. Plusieurs études ont montré que la metformine et le glyburide sont deux alternatives acceptables en grossesse. Malgré tout, l’insuline reste le choix de première intention pour le traitement du DG lorsque la diète et l’exercice physique ne suffisent plus. Les principaux arguments en faveur des HGO sont la possibilité d’une meilleure observance du traitement et une meilleure qualité de vie (QDV). Cependant, les études sur la QDV chez les femmes atteintes de DG sont rares et d’autres études sur le HGO sont nécessaires pour valider leur efficacité et innocuité dans le traitement du DG. Méthodes : Étude pilote; 73 patientes avec DG ont été randomisées au groupe HGO (metformine ± glyburide, et ajout d’insuline au besoin) ou au groupe insuline. L’objectif primaire est de comparer, entre les deux groupes, la préférence du traitement ainsi que plusieurs aspects de la QDV (état de santé général, bien-être général, satisfaction quant au traitement du diabète et dépression post-natale). Les objectifs secondaires étaient de comparer le contrôle glycémique maternel et les issues maternelles et néonatales. Résultats : Des 73 femmes recrutées, 68 ont été incluses pour analyses. Dans le groupe HGO (n=34), 35,3% des participantes ont reçu de l’insuline. Plus de femmes dans le groupe HGO ont mentionné préférer recevoir le même traitement pour une prochaine grossesse (72.7% vs. 42,4%, p=0.01) si un traitement s’avérait nécessaire. Aucune différence n’est ressortie pour l’état de santé général, le bien-être général, la satisfaction quant au traitement et la dépression post-natale. Le taux moyen d’hypoglycémies maternelles (glycémie < 3.3 mmol/L) était supérieur dans le groupe HGO (0.8 vs. 0.1, p=0.008). Il n’y a eu aucune différence dans les autres complications maternelles et néonatales. Conclusion : La QDV est similaire dans les deux groupes mais, si un traitement pharmacologique s’avérait nécessaire lors d’une prochaine grossesse, les femmes préfèreraient débuter par les HGO. L’utilisation des HGO n’était pas associée à une hausse cliniquement significative des complications maternelles et néonatales. / Abstract: Background: The use of oral hypoglycemic agents (OHA) in gestational diabetes mellitus (GDM) is still debated. Insulin remains the first line treatment after diet failure. We hypothesized that OHA allows for better quality of life (QOL) in women with GDM and that the y prefer OHA to insulin therapy. Methods: Pilot study; 73 women with GDM were randomly assigned to the OHA group (metformin ± glyburide ± supplementary insulin, as needed) or the insulin group. General health, well-being, and treatment satisfaction using established questionnaires (SF-36, Well-Being Questionnaire and Diabetes Treatment Satisfaction Questionnaire) were respectively assessed at randomization, 38 weeks, and postpartum. Treatment preference and depression were assessed 8 weeks postpartum using the MIG questionnaire and the Short-form Edinburgh Depression Scale. Our primary outcomes were 1) preference of treatment and 2) QOL. Secondary outcomes included maternal glycemic control (hypoglycemia defined as plasma glucose < 3.3 mmol/L), as well as gestational and neonatal complications. Results: Analyses were performed in 68 women. In the OHA group (n= 34), 35.3% women received insulin therapy. More women in the OHA group than in the insulin group preferred their assigned treatment for another pregnancy (72.7% vs. 42.4%, p < 0.01) if a treatment is needed. No statistical differences between groups were found for the 3 items of QOL, the depression score, and neonatal complications. The mean rate of maternal hypoglycemia was higher in the OHA group (0. 8 vs. 0.1, p < 0.008). No statistical differences were found for other maternal and neonatal issues. Conclusion: Women declared to prefer OHA to insulin for a subsequent pregnancy complicated by GDM. QOL was similar in both groups. Use of OHA was not associated to clinically significant differences in maternal and neonatal complications.

As plantas medicinais têm sido utilizadas como uma importante fonte de prevenção e tratamento de doenças para a população pobre dos países em desenvolvimento. A literatura apresenta resultados que demonstram a atividade de proteínas e flavonóides presentes nas folhas, sementes e cascas do caule de Bauhinia forficata Link com ação hipoglicemiante. Este trabalho tem como objetivo estabelecer um protocolo para a obtenção das células em cultura de B. forficata para posterior produção de compostos fenólicos e proteínas com atividade hipoglicemiante. Assim, inicialmente, foram padronizadas as condições para obtenção da cultura in vitro de B. forficata Link. Realizou-se a indução da calogênese (indução máxima 77%) utilizando-se folhas como explantes em meio MS suplementado com ácido 2,4-diclofenoxiacético 1 mg/L, cinetina 1 mg/L e 3% (m/v) de sacarose. A cultura de células em suspensão foi estabelecida a partir da transferência de inóculos de calos frescos (2g) para a 40 mL de meio MS. Após a padronização realizou-se elicitações, com MeJA e AS 1 mol/L, nos tempo de 0, 3, 6, 9 e 12 dias, monitoradas pelo teor de compostos fenólicos e proteínas totais no meio intra e extracelular. Avaliou-se também o efeito da suplementação com sacarose (20, 30 e 40 g/L) sobre o sistema celular. O MeJA causou um aumento de composto fenólicos no meio extracelular de 105% e de 126% no meio intracelular, em 6 dias. O AS, em 9 dias de indução, aumentou esse compostos em 106,5% e 132,6% no meio extra e intracelular, respectivamente. Em 6 dias obteve-se o melhor tempo de elicitação das proteínas extracelulares com MeJA (81%) e de 9 dias para AS (41%). A presença desses elicitores, em 6 e 9 dias, foram capazes de aumentar a atividade das enzimas fenilalanina amônia liase (FAL) e tirosina amônia liase (TAL) no meio intracelular. Os resultados obtidos demonstraram que AS e MeJA podem modular a liberação de compostos fenólicos e proteínas para o meio extracelular, sendo os tempos de 6 e 9 dias os mais eficientes. A suplementação do meio com sacarose contribui para esta liberação, sendo a melhor concentração de sacarose 30 g/L. Portanto, as condições estabelecidas foram eficientes para obtenção da suspensão celular de Bauhinia forficata Link e modulação de compostos fenólicos e proteínas neste sistema. / The medicinal plants have been used as a major source of prevention and treatment of diseases for the poor population in the developing countries. Hypoglycaemic bioactive compounds have been found in leaves, seeds and barks of Bauhinia forficata. The aim for this work was to establish a protocol to obtain the B. forficata cell culture and to product phenolic compounds and proteins with hypoglycaemic activities. The callogenesis induction (maximum response: 77%) was developed using leaves as explants on MS medium supplemented with 1mg/L 2,4-diclorophenoxiacetic acid (2,4-D) and 1mg/L kinetin plus 3% sucrose (w/v). The cell suspension culture was established by transferring an inoculum of fresh callus (2 g) to 40 mL liquid MS medium. The effects of 1 mol/L methyl jasmonate (MeJA) or salicylic acid (AS) in the cell culture, supplemented with sucrose (20, 30 and 40 g/L), was evaluated by monitoring the proteins and phenolic compounds in the extra and intra cellular medium after 0, 3, 6, 9 and 12 days. In the extracellular fractions, the best time for phenolic compounds induction in the presence of MeJA (105%) and AS (106.5%) was 6 and 9 days, respectively. In the intracellular fractions, the highest effect was observed 6 days after induction in the presence of MeJA (126%) or AS (132.6%). The best time for protein induction in the extracellular medium was 6 days in the presence of MeJA (81%) and 9 days in the presence of AS (41%). In addition, the activity of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) increased in the intracellular medium in the presence of MeJA and AS after 6 and 9 days, respectively. In conclusion, the conditions we used were efficient for obtention of cell suspension culture of B. forficata. MeJA and AS can be used as elicitors of phenolic compounds and protein in this cellular system.

As plantas medicinais têm sido utilizadas como uma importante fonte de prevenção e tratamento de doenças para a população pobre dos países em desenvolvimento. A literatura apresenta resultados que demonstram a atividade de proteínas e flavonóides presentes nas folhas, sementes e cascas do caule de Bauhinia forficata Link com ação hipoglicemiante. Este trabalho tem como objetivo estabelecer um protocolo para a obtenção das células em cultura de B. forficata para posterior produção de compostos fenólicos e proteínas com atividade hipoglicemiante. Assim, inicialmente, foram padronizadas as condições para obtenção da cultura in vitro de B. forficata Link. Realizou-se a indução da calogênese (indução máxima 77%) utilizando-se folhas como explantes em meio MS suplementado com ácido 2,4-diclofenoxiacético 1 mg/L, cinetina 1 mg/L e 3% (m/v) de sacarose. A cultura de células em suspensão foi estabelecida a partir da transferência de inóculos de calos frescos (2g) para a 40 mL de meio MS. Após a padronização realizou-se elicitações, com MeJA e AS 1 mol/L, nos tempo de 0, 3, 6, 9 e 12 dias, monitoradas pelo teor de compostos fenólicos e proteínas totais no meio intra e extracelular. Avaliou-se também o efeito da suplementação com sacarose (20, 30 e 40 g/L) sobre o sistema celular. O MeJA causou um aumento de composto fenólicos no meio extracelular de 105% e de 126% no meio intracelular, em 6 dias. O AS, em 9 dias de indução, aumentou esse compostos em 106,5% e 132,6% no meio extra e intracelular, respectivamente. Em 6 dias obteve-se o melhor tempo de elicitação das proteínas extracelulares com MeJA (81%) e de 9 dias para AS (41%). A presença desses elicitores, em 6 e 9 dias, foram capazes de aumentar a atividade das enzimas fenilalanina amônia liase (FAL) e tirosina amônia liase (TAL) no meio intracelular. Os resultados obtidos demonstraram que AS e MeJA podem modular a liberação de compostos fenólicos e proteínas para o meio extracelular, sendo os tempos de 6 e 9 dias os mais eficientes. A suplementação do meio com sacarose contribui para esta liberação, sendo a melhor concentração de sacarose 30 g/L. Portanto, as condições estabelecidas foram eficientes para obtenção da suspensão celular de Bauhinia forficata Link e modulação de compostos fenólicos e proteínas neste sistema. / The medicinal plants have been used as a major source of prevention and treatment of diseases for the poor population in the developing countries. Hypoglycaemic bioactive compounds have been found in leaves, seeds and barks of Bauhinia forficata. The aim for this work was to establish a protocol to obtain the B. forficata cell culture and to product phenolic compounds and proteins with hypoglycaemic activities. The callogenesis induction (maximum response: 77%) was developed using leaves as explants on MS medium supplemented with 1mg/L 2,4-diclorophenoxiacetic acid (2,4-D) and 1mg/L kinetin plus 3% sucrose (w/v). The cell suspension culture was established by transferring an inoculum of fresh callus (2 g) to 40 mL liquid MS medium. The effects of 1 mol/L methyl jasmonate (MeJA) or salicylic acid (AS) in the cell culture, supplemented with sucrose (20, 30 and 40 g/L), was evaluated by monitoring the proteins and phenolic compounds in the extra and intra cellular medium after 0, 3, 6, 9 and 12 days. In the extracellular fractions, the best time for phenolic compounds induction in the presence of MeJA (105%) and AS (106.5%) was 6 and 9 days, respectively. In the intracellular fractions, the highest effect was observed 6 days after induction in the presence of MeJA (126%) or AS (132.6%). The best time for protein induction in the extracellular medium was 6 days in the presence of MeJA (81%) and 9 days in the presence of AS (41%). In addition, the activity of phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) increased in the intracellular medium in the presence of MeJA and AS after 6 and 9 days, respectively. In conclusion, the conditions we used were efficient for obtention of cell suspension culture of B. forficata. MeJA and AS can be used as elicitors of phenolic compounds and protein in this cellular system.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.